The balance of success in cancer diagnosis

Recent studies have suggested that there is no “magic bullet” for diagnosis of cancer and other similarly complex diseases. Instead, many believe, successful diagnosis may depend on identifying a profile of various protein markers. For this reason, researchers are working to develop multiplexed platforms with which to screen large numbers of markers for such “diagnostic profiles” of cancers.

In the past several years, researchers with the University of California, Berkeley, with the University of Southern California, Los Angeles, and with Lawrence Berkeley National Laboratory, also in Berkeley, led by Arun Majumdar, have reported DNA hybridization studies using two-dimensional microcantilever arrays — which, they note, can be easily scaled up for high-throughput studies with hundreds of simultaneous reactions. In a February Nano Letters paper, they described a label-free immunoassay using a two-dimensional microcantilever array to screen for diagnostic profiles of cancer and other diseases.

Investigators have developed a label-free immunoassay that can be used to screen for diagnostic profiles of cancer and other diseases. The platform is based on a two-dimensional microcantilever array. Any biological reactions that take place on the surface of a cantilever will lead to changes in the surface stress. This in turn will cause deflection of the cantilever, which will show the researchers whether a reaction has occurred. Shown here is a single reaction cell/chamber — array chips contain 80 to 120 of these — with six cantilevers.Microcantilever arrays can be used for an immunoassay because biological reactions occurring on the surface of a cantilever will induce changes in the surface stress, leading to deflection of the cantilever, which can be monitored and used to determine whether a reaction has occurred. In the present study, a unique chip design and an effective surface chemistry enabled antibody-antigen detection on an array, thus underscoring the diagnostic potential of the technique.

The array chips included 80 to 120 reaction wells, each of which consisted of a microfluidic chamber with four to eight cantilever sensors. The cantilevers themselves were 0.5 μm thick, 40 μm wide and 200 to 400 μm long. One surface was coated with a 25-nm gold layer for immobilization of antibodies. The investigators illuminated this surface from the glass side with a collimated beam from a laser made by Coherent Inc. of Santa Clara, Calif., expanding the spot to roughly the size of the array, about 2.5 cm in diameter. A CCD camera made by Apogee Inc. of Northbrook, Ill., detected the light reflecting off the cantilevers’ end pads, imaging it as an array of “spots.” These light spots revealed the state of the corresponding cantilever’s deflection, thus indicating to the researchers whether binding had occurred.

Developing the microcantilever array for this particular application proved to be a challenge. In addition to coming up with the most appropriate chip design, the researchers had to address the question of surface chemistry.

“Cantilever detection is very unique, so some known chemistries did not work well with it,” explained Min Yue, the first author of the study. “We did not limit ourselves and tried different combinations of functionalizations and passivations.”

They tested three different cross-linkers for immobilizing antibodies on the surface — DTSSP, NHS-thiol and biotin-NeutrAvidin — and explored passivation by both BSA and PEG-silane. They found that the cantilever response to antibody-antigen binding was consistent only when they used DTSSP or NHS-thiol as a cross-linker and PEG-silane as a passivation agent.

Using these chemistries, therefore, they measured the cantilevers’ responses to binding of functionalized prostate specific antigen (PSA) to an anti-PSA antibody immobilized on the surface, for different concentrations of PSA. They found, generally, that decreases in antigen concentration led to decreases in the surface stress changes caused by binding on the cantilevers. In addition, they noted that they could detect concentrations of PSA as low as 1 ng/ml. Thus the arrays could form the basis of real-time, label-free, multiplexed immunoassays with which to perform high-sensitivity detection of diagnostic profiles for cancer.

The researchers pointed to several advantages with respect to other methods available for detection of molecular bindings. Compared with fluorescence methods, for example, the label-free detection offered by the cantilever platform is “a huge advantage,” Yue said. He noted that, if they were to commercialize the technique, the chip would be more expensive than the simple glass slide used in fluorescence detection. “However, keep in mind that the labeling process in fluorescence detection demands more labor and reagents.”

Compared with other cantilever platforms based on optical detection, he continued, the 2-D microarray system is easily scaled up, and the readout system is very simple; previous studies offered only one-dimensional arrays and required a more complicated optics hardware setup. “There are two-dimensional piezoresistive cantilever arrays. Even though they don’t require optical setup, the chip fabrication is more complicated, so the chips may be more expensive.”

The researchers plan to apply the technique to the study of enzymatic activity as well as to small-molecule detection in liquids and gaseous environments.